Cells with neuroendocrine (NE)-Iike properties have been implicated in progression of prostate cancer to hormone independence and increased aggressiveness. NE-like cells are postulated to promote the survival, growth, and metastatic capabilities of surrounding tumor cells by secreting factors that promote these processes, particularly in an androgen-depleted environment. In a nude mouse xenograft model, we tested this paracrine hypothesis and showed that artificially engineered NE-like LNCaP cells provide a growth advantage to non-engineered LNCaP tumor cells following castration (androgen ablation), as compared to LNCaP tumor cells lacking such NE cells upon inoculation. However, the mechanism of action of the NE-like cells in this assay is unknown, i.e., whether they promote mitogenesis or survival, or whether they enhance selection of already existing genetic variants within a population of tumor cells that exhibit a more aggressive and androgen-independent phenotype. It has also not been tested in vivo whether these cells secrete factors that promote metastases. We propose to study these various possibilities in the nude mouse model we have established by examining tumors that arise following castration from various combinations of NE-like cells and parental LNCaP cells for markers of apoptosis (caspase activation, TUNEL, annexin V, etc.), proliferation (Ki67, MAP kinase activation, STAT tyrosine phosphorylation, etc.), metastases, or ability to exhibit enhanced growth following re-implantation either with or without castration. In addition, we will investigate the signaling pathways stimulated by the secreted products of NE-like cells. Most of the known secretory products of NE-like cells are agonists for G protein coupled receptors (GPCRs). A growing literature indicates that many GPCRs transactivate and require the EGF receptor (EGFR) (specifically phosphorylation of Tyr 845 on the EGFR by c-Src) for their action. Based on these findings and the fact that the EGFR has been implicated as an etiological agent in prostate cancer, we will test the dependence on the EGFR of GPCR agonists secreted by NE-like cells for inducing survival, growth and/or metastatsis of prostate cancer cells. We also propose strategies to identify downstream effectors of EGFR and Tyr 845 and to determine effects of Tyr 845-containing inhibitory peptides on prostate cancer growth. This approach of targeting a widely used and critical EGFR phosphorylation site (Tyr 845) is hypothesized to provide a novel means of counteracting the action of NE-like cells in promoting aggressive growth of androgen-independent prostate cancers.